Inhibition Of Clove Essential Oil On Literia Monocytogeness And Its Biofilm

Listeria monocytogenes(L.monocytogenes)is widely distributed in nature and is one of the most threatening food-borne pathogens announced by WHO.L.monocytogenes can adhere to food processing equipment and storage containers.After forming a biofilm,they can contaminate food through dispersion and cause serious food safety problems.Therefore,in this paper,L.monocytogenes was selected as the main test bacteria,and the antibacterial effect and mechanism of natural antibacterial agent Clove essential oil(CEO)on its free bacteria and biofilms were studied in depth.Research on the antibacterial mechanism of free bacteria includes the antibacterial activity of essential oils against L.monocytogenes,the effects on the content of three intracellular macromolecular substances and the activity of two key intracellular enzymes,the main inhibitory pathways on respiratory metabolism,as well as interaction between eugenol and DNA.On this basis,the mechanism of CEO's inhibition of biofilms was further explored,and the effects of CEO on several key regulators of biofilm formation were measured,including early cell motility,extracellular matrix(protein,polysaccharide,DNA)secretion and expression levels of L.monocytogenes biofilm formation related genes agr operon genes,virulence regulator prfA and stress response factor sigB gene.Finally,the CEO was applied to the surface of vegetables and different container materials,and the actual application effect was evaluated.The main findings are as follows:(1)CEO has significant antibacterial activity against L.monocytogenes.The MIC and MBC are 0.5 mg/mL and 1.0 mg/mL,respectively,and the bactericidal rate is 99.999% after 8 h treated with MIC concentration.TEM results showed that after the action of essential oils,folds and depressions appeared on the surface of the bacterial cells,and the cell membrane ruptured,leading to the leakage of cell contents.Further verification revealed that after the action of essential oils,the content of three intracellular macromolecular substances(protein,ATP,DNA)and the activity of two key intracellular enzymes(?-galactosidase and AKP enzyme)were inhibited.(2)The chief pathway by which CEO inhibits respiratory metabolism of L.monocytogene is the TCA pathway.The activities of the three key enzymes(CS,?-KGDH,ICDHm)in the TCA pathway were inhibited by the essential oils.Analysis of the changes in the content of metabolites related to the TCA cycle shows that the TCA cycle weakens after the action of essential oils.In addition,CEO's main component eugenol can be chimeric with bacterial DNA.(3)CEO has good scavenging effect on L.monocytogenes biofilm.The MBIC and MBEC are 1.0 mg/mL and 2.0 mg/mL,respectively,and after 8 h of treatment with 4MBIC concentration of essential oil,the biofilm removal rate reached 99.99%.TEM and CLSM observed that the essential oil would destroy the dense membrane structure of the bacteria,thereby acting on the released free bacteria to reduce their activity or even inactivate them.(4)CEO inhibits the metabolic activity of bacteria in the biofilm.After treated with 2MBIC concentration of essential oil,60.26% of the bacteria in the biofilm lost their metabolic activity.After the CEO's action,the bacteria's ability to aggregate is reduced,and the range of cluster movement is reduced,thereby inhibiting the initial stage of biofilm formation.(5)In the extracellular polymer of L.monocytogenes EDG-e biofilm,the contents of protein,DNA and polysaccharide decrease sequentially.After treatment with 2MBIC concentration of essential oil,the extracellular polysaccharide and protein secretion decreased by 88.34% and 75.61%,respectively,but secretion of DNA did not change significantly.(6)CEO influences the expression of genes related to L.monocytogenes EDG-e biofilm formation.After the effect of MIC concentration of essential oil,the expression levels of agrA and agrC decreased by 92.44% and 90.18%,respectively,while the expression level of agrB gene did not change significantly,and the expression level of sigB gene was increased by 2.37 times.The expression of gene prfA was reduced by 84.33%.(7)Applied research shows,the CEO showed significant removal effect on L.monocytogenes biofilms on different container materials and vegetable surfaces,and sensory evaluation experiments showed that essential oils did not destroy the quality and original flavor of vegetables.